Human amnion cell culture: current limitations and potential for regenerative medicine

Aims: The identification of human amnion cells capable to acquire different cell fates in vitro, has provided exciting prospects for cell-based regenerative medicine. We focus on side-by-side analysis of amnion epithelial (AEC) and mesenchymal cell (AMC) for their vitality, expandibility and ‘stemness profilersquor; after isolation and under culture.

Materials and Methods: AEC and AMC were isolated from 27 term placentas. Cell number, morphology, size, proliferation and apoptosis were analysed. Cells were characterized by flow cytometry, immunocytochemistry and RT-PCR for expression of immunologic and stem cell (SC)-markers.

Results: Primary yield of AEC and AMC was 6×106 and 1.5×106 per gram amnion, respectively. All 27 cases gave adherent vital cultures of AMC, while one third of AEC isolates (9 of 27) failed to adhere. Primary cultures contained significantly more proliferating than apoptotic cells. Neither AEC nor AMC were clonogenic. They showed slow proliferation that almost stopped beyond passage 5. AEC morphology changed towards mesenchymal phenotype over several passages. Flow cytometric characterization showed expression of mesenchymal progenitor markers CD73, CD90, CD105 and CD166 and the embryonic SC markers STRO–1, SSEA–3 and –4 on both cell types; levels of SSEA–4 markedly decreased during subculture. AEC and AMC expressed high levels of HLA-ABC but no HLA-DR. RT-PCR analyses exhibited transcripts of Oct–3/4 and stem cell factor, but no of telomerase reverse transcriptase.

Conclusion: AEC and AMC in culture exhibit a similar marker profile of mesenchymal progenitors. AEC were found as less reliable source than AMC and altered morphology during subculture.